GSM6685484: tissue, tumor, patient 9; Homo sapiens; RNA-Seq
The samples were taken out at -80 ℃ and immediately put into a liquid nitrogen precooled mortar. After adding liquid nitrogen to grind into powder, 50mg samples were weighed, 1ml TRizol was added, vortex mixing was carried out, 10min was placed at room temperature, 200ul chloroform was added, mixing was fully reversed for 15s, and 2-3min was placed at room temperature. 4 ℃, 12000rpm centrifuge 10min (pay attention to pre-cooling centrifuge), carefully absorb the supernatant of 400ul into a new EP tube, add isopropanol at 1:1 (V400ul V), vibrate and mix 15s 10min at 20 ℃, place 10min at 4 ℃, 12000rpm centrifuge 10min, discard the supernatant, wash the precipitation with 75% ethanol twice, centrifuge 1min at 4 ℃, thoroughly remove the supernatant, dry the centrifuge tube in a super clean table to transparent, add 40ul RNase-free water to dissolve. The concentration of the sample and the ratio of 260 to 280260 to 230 were measured and recorded by Nanodrop. The integrity of RNA was tested by Agilent 4150, and the sample was preserved at-80 ℃. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size -selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 ° C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Illumina Novaseq PE150.